Abstract 1928: Impaired Endocytosis of TRPM4 Channel Associated With Progressive Familial Heart Block Type I
Progressive familial heart block type I (PFHBI) is a dominantly inherited cardiac bundle-branch disorder that has previously been mapped to chromosome 19q13. Here we show that a mutation in the gene encoding for the ion channel TRPM4 correlates with the occurrence of PFHBI. Genetic analysis of members of a large South African pedigree affected by PFHBI revealed a mutation in the TRPM4 gene (c.19G>A), predicting a substitution of glutamic acid at position 7 to lysine (p.E7K) within the TRPM4 N-terminus. Genes for other cardiac ion channels were not altered. Quantitative analysis of TRPM4-mRNA content in human cardiac tissue showed highest expression level in Purkinje fibers. Whole-cell patch-clamp recordings from HEK293-cells transiently transfected with mutated TRPM4-channel showed a significant gain-of-function associated with an increase in TRPM4 current density (TRPM4: 18.8 ± 3.7 pA/pF; TRPM4-E7K: 43.8 ± 6.3 pA/pF; n > 10). Coexpression with TRPM4-wt channel displayed a dominant phenotype of mutated channel. Ca2+-dependence, single channel conductance and open probability remained unaffected in the mutant channel in comparison to wild-type. The data indicated that the observed mutational effect on TRPM4-current amplitude was most likely due to a change in channel surface density. In order to evaluate TRPM4 channel surface density, we introduced a Myc-tag into an extracellular site of TRPM4 for quantitative analysis of TRPM4 surface density using fluorescence-activated cell-sorting (FACS). In agreement with our electrophysiological results, FACS-analysis showed for Myc-tagged TRPM4-E7K a ~ two-and-a-half fold larger surface density than for wild-type. Additionally, we found an increased steady-state concentration of mutated protein in Western-blots. Cellular expression studies showed that the mutation interferes with dynein-dependent endocytosis of TRPM4 channel. We propose that increased cardiac TRPM4-channel activity slows or even blunts cardiac conduction because of its effects on membrane depolarization and Ca2+ influx. The influence of expression of mutant TRPM4-channels in primary cardiac cells is under investigation.